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Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head.

Nakata T, Hirokawa N - J. Cell Biol. (2003)

Bottom Line: Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons.We found that KIF5 has a preference to the microtubules in the initial segment of axon.These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Tokyo, Japan 113-0033.

ABSTRACT
Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1-YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

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KIF5 is preferentially recruited to the MTs in the IS. After brief expression of rigor mutants of motor proteins, cells were permeabilized, fixed, and stained with antibodies. CLSM images of the strongly bound motors were obtained by serial Z-sectioning with a small confocal aperture to avoid the effect of the thickness of the cells. Data are presented as a maximal Z-projection image. Arrows indicate the IS. (a and b) Neurons expressing tailless KIF5B G234A::YFP (green) were stained with anti-MAP2 antibody (a, red) or with anti–β-tubulin antibody (b, red). Center of the cell body and the IS were intensely labeled by rigor-KIF5. (c and d) Neurons expressing tailless KIF17 G243A::YFP (green) were stained with anti-MAP2 antibody (c, red) and with anti–β-tubulin antibody (d, red). Dendrites as well as the IS (c, arrow) were intensely labeled with rigor-KIF17. (e) Simultaneous double-labeling with rigor-KIF5 (red) and tailless rigor-KIF17::YFP (green). KIF5 was stained with antikinesin antibody H2. Although the IS was intensely labeled by the both motors (arrow), KIF5 preferentially labels the center of the cell body, whereas KIF17 labels dendrites as well as axons. (f) Simultaneous double-labeling with rigor-KIF5 (green) and rigor-KIF2 (red). Although the labeling pattern in the cell body was similar in both KIFs, KIF5 preferentially labels the IS (arrow), whereas rigor-KIF2 does not. Bars, 10 μm. (g–i) Immuno-electron microscopy of rigor-KIF5 in hippocampal neurons. Rigor-KIF5 was labeled with 5-nm colloidal gold followed by silver enhancement. (g) Intense labeling of the IS is observable even at this low magnification (arrow). C indicates the cell body. (h) Higher magnification of the dendrite area in g (box D). (i) Higher magnification of axon area in g (box A). Note that extensive labeling of axons compared with dendrites. Bar, 500 nm.
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fig4: KIF5 is preferentially recruited to the MTs in the IS. After brief expression of rigor mutants of motor proteins, cells were permeabilized, fixed, and stained with antibodies. CLSM images of the strongly bound motors were obtained by serial Z-sectioning with a small confocal aperture to avoid the effect of the thickness of the cells. Data are presented as a maximal Z-projection image. Arrows indicate the IS. (a and b) Neurons expressing tailless KIF5B G234A::YFP (green) were stained with anti-MAP2 antibody (a, red) or with anti–β-tubulin antibody (b, red). Center of the cell body and the IS were intensely labeled by rigor-KIF5. (c and d) Neurons expressing tailless KIF17 G243A::YFP (green) were stained with anti-MAP2 antibody (c, red) and with anti–β-tubulin antibody (d, red). Dendrites as well as the IS (c, arrow) were intensely labeled with rigor-KIF17. (e) Simultaneous double-labeling with rigor-KIF5 (red) and tailless rigor-KIF17::YFP (green). KIF5 was stained with antikinesin antibody H2. Although the IS was intensely labeled by the both motors (arrow), KIF5 preferentially labels the center of the cell body, whereas KIF17 labels dendrites as well as axons. (f) Simultaneous double-labeling with rigor-KIF5 (green) and rigor-KIF2 (red). Although the labeling pattern in the cell body was similar in both KIFs, KIF5 preferentially labels the IS (arrow), whereas rigor-KIF2 does not. Bars, 10 μm. (g–i) Immuno-electron microscopy of rigor-KIF5 in hippocampal neurons. Rigor-KIF5 was labeled with 5-nm colloidal gold followed by silver enhancement. (g) Intense labeling of the IS is observable even at this low magnification (arrow). C indicates the cell body. (h) Higher magnification of the dendrite area in g (box D). (i) Higher magnification of axon area in g (box A). Note that extensive labeling of axons compared with dendrites. Bar, 500 nm.

Mentions: Next, we addressed the question whether KIF5 is activated preferentially between TGN and axons. To test it, motor proteins that can be recruited to MTs but cannot translocate along nor dissociate from MTs are suitable because such mutant motor proteins will show the site of initial recruitment of the motor proteins by their localization. We know of three such point mutations of kinesin (rigor kinesins) that are well characterized (Nakata and Hirokawa, 1995; Rice et al., 1999). We found that all the mutations (T92N, G234A, and E236A) show the same results in the case of KIF5B. KIF5 G234A mutant and corresponding KIF17 G243A mutant were localized on MTs in transfected cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). We expressed rigor-motor proteins fused with YFP in hippocampal neurons, permeabilized them in order to detect only MT-bound fractions. CLSM enabled us to compare the rigor-motor binding eliminating the effect of the difference in the thickness of axons and somatodendrites. We found that rigor-KIF5 with G234A mutation distributed most intensely on MTs from the center of the cell body to the initial segment of axon (IS), which is confirmed by MAP2 staining (Fig. 4 a; fluorescence ratios of rigor-KIF5::GFP axon to dendrites and dendrites to cell body were 217 ± 81.1% and 63.0 ± 20.8%, respectively [n = 25]). Double labeling with tubulin demonstrated relatively low density association of rigor-KIF5 with MTs in dendrites and high density association in the center of the cell body and the IS (Fig. 4 b; fluorescence ratios of rigor-KIF5::GFP to tubulin staining was 1.2 ± 0.4 [n = 14] in axons, and 0.5 ± 0.2 [n = 22] in dendrites). In contrast, rigor-KIF17 with corresponding G243 mutation distributed on MTs in both dendrites and the IS (Fig. 4 c). Double labeling with tubulin demonstrated high density association of the rigor-KIF17 with MTs in dendrites and the IS (Fig. 4 d). In the cell body, the rigor-KIF17 binds strongly to some MTs, but they show lower affinity with MTs in other areas of the cell body (fluorescence ratio of rigor-KIF17::YFP in axon to dendrites and dendrites to cell body were 109 ± 28.0% and 402 ± 281%, respectively [n = 25]). Simultaneous expression of rigor-KIF5 and rigor-KIF17 indicates that the preference of these two types is clearly different within the same neurons (Fig. 4 e). This distribution of rigor-KIF5 and -KIF17 is consistent with the destinations of tailless motors. Although motor domains of KIF5 and KIF17 had a property to bind to MTs in the IS, this is not always the case because rigor-KIF2 did not localize on MTs in the IS, as shown by the simultaneous expression of rigor-KIF2 and rigor-KIF5 in hippocampal neurons (Fig. 4 f). We further examined the axon preference of the rigor-KIF5 by using immunoelectron microscopy. Fig. 4 (g–i) shows biased binding of rigor-KIF5 to the IS (Fig. 4 g, arrow). These results suggest that KIF5 motor domain, as default, has a preference to the MTs in the IS and tailless KIF5 is preferentially sorted to axons.


Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head.

Nakata T, Hirokawa N - J. Cell Biol. (2003)

KIF5 is preferentially recruited to the MTs in the IS. After brief expression of rigor mutants of motor proteins, cells were permeabilized, fixed, and stained with antibodies. CLSM images of the strongly bound motors were obtained by serial Z-sectioning with a small confocal aperture to avoid the effect of the thickness of the cells. Data are presented as a maximal Z-projection image. Arrows indicate the IS. (a and b) Neurons expressing tailless KIF5B G234A::YFP (green) were stained with anti-MAP2 antibody (a, red) or with anti–β-tubulin antibody (b, red). Center of the cell body and the IS were intensely labeled by rigor-KIF5. (c and d) Neurons expressing tailless KIF17 G243A::YFP (green) were stained with anti-MAP2 antibody (c, red) and with anti–β-tubulin antibody (d, red). Dendrites as well as the IS (c, arrow) were intensely labeled with rigor-KIF17. (e) Simultaneous double-labeling with rigor-KIF5 (red) and tailless rigor-KIF17::YFP (green). KIF5 was stained with antikinesin antibody H2. Although the IS was intensely labeled by the both motors (arrow), KIF5 preferentially labels the center of the cell body, whereas KIF17 labels dendrites as well as axons. (f) Simultaneous double-labeling with rigor-KIF5 (green) and rigor-KIF2 (red). Although the labeling pattern in the cell body was similar in both KIFs, KIF5 preferentially labels the IS (arrow), whereas rigor-KIF2 does not. Bars, 10 μm. (g–i) Immuno-electron microscopy of rigor-KIF5 in hippocampal neurons. Rigor-KIF5 was labeled with 5-nm colloidal gold followed by silver enhancement. (g) Intense labeling of the IS is observable even at this low magnification (arrow). C indicates the cell body. (h) Higher magnification of the dendrite area in g (box D). (i) Higher magnification of axon area in g (box A). Note that extensive labeling of axons compared with dendrites. Bar, 500 nm.
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Related In: Results  -  Collection

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fig4: KIF5 is preferentially recruited to the MTs in the IS. After brief expression of rigor mutants of motor proteins, cells were permeabilized, fixed, and stained with antibodies. CLSM images of the strongly bound motors were obtained by serial Z-sectioning with a small confocal aperture to avoid the effect of the thickness of the cells. Data are presented as a maximal Z-projection image. Arrows indicate the IS. (a and b) Neurons expressing tailless KIF5B G234A::YFP (green) were stained with anti-MAP2 antibody (a, red) or with anti–β-tubulin antibody (b, red). Center of the cell body and the IS were intensely labeled by rigor-KIF5. (c and d) Neurons expressing tailless KIF17 G243A::YFP (green) were stained with anti-MAP2 antibody (c, red) and with anti–β-tubulin antibody (d, red). Dendrites as well as the IS (c, arrow) were intensely labeled with rigor-KIF17. (e) Simultaneous double-labeling with rigor-KIF5 (red) and tailless rigor-KIF17::YFP (green). KIF5 was stained with antikinesin antibody H2. Although the IS was intensely labeled by the both motors (arrow), KIF5 preferentially labels the center of the cell body, whereas KIF17 labels dendrites as well as axons. (f) Simultaneous double-labeling with rigor-KIF5 (green) and rigor-KIF2 (red). Although the labeling pattern in the cell body was similar in both KIFs, KIF5 preferentially labels the IS (arrow), whereas rigor-KIF2 does not. Bars, 10 μm. (g–i) Immuno-electron microscopy of rigor-KIF5 in hippocampal neurons. Rigor-KIF5 was labeled with 5-nm colloidal gold followed by silver enhancement. (g) Intense labeling of the IS is observable even at this low magnification (arrow). C indicates the cell body. (h) Higher magnification of the dendrite area in g (box D). (i) Higher magnification of axon area in g (box A). Note that extensive labeling of axons compared with dendrites. Bar, 500 nm.
Mentions: Next, we addressed the question whether KIF5 is activated preferentially between TGN and axons. To test it, motor proteins that can be recruited to MTs but cannot translocate along nor dissociate from MTs are suitable because such mutant motor proteins will show the site of initial recruitment of the motor proteins by their localization. We know of three such point mutations of kinesin (rigor kinesins) that are well characterized (Nakata and Hirokawa, 1995; Rice et al., 1999). We found that all the mutations (T92N, G234A, and E236A) show the same results in the case of KIF5B. KIF5 G234A mutant and corresponding KIF17 G243A mutant were localized on MTs in transfected cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). We expressed rigor-motor proteins fused with YFP in hippocampal neurons, permeabilized them in order to detect only MT-bound fractions. CLSM enabled us to compare the rigor-motor binding eliminating the effect of the difference in the thickness of axons and somatodendrites. We found that rigor-KIF5 with G234A mutation distributed most intensely on MTs from the center of the cell body to the initial segment of axon (IS), which is confirmed by MAP2 staining (Fig. 4 a; fluorescence ratios of rigor-KIF5::GFP axon to dendrites and dendrites to cell body were 217 ± 81.1% and 63.0 ± 20.8%, respectively [n = 25]). Double labeling with tubulin demonstrated relatively low density association of rigor-KIF5 with MTs in dendrites and high density association in the center of the cell body and the IS (Fig. 4 b; fluorescence ratios of rigor-KIF5::GFP to tubulin staining was 1.2 ± 0.4 [n = 14] in axons, and 0.5 ± 0.2 [n = 22] in dendrites). In contrast, rigor-KIF17 with corresponding G243 mutation distributed on MTs in both dendrites and the IS (Fig. 4 c). Double labeling with tubulin demonstrated high density association of the rigor-KIF17 with MTs in dendrites and the IS (Fig. 4 d). In the cell body, the rigor-KIF17 binds strongly to some MTs, but they show lower affinity with MTs in other areas of the cell body (fluorescence ratio of rigor-KIF17::YFP in axon to dendrites and dendrites to cell body were 109 ± 28.0% and 402 ± 281%, respectively [n = 25]). Simultaneous expression of rigor-KIF5 and rigor-KIF17 indicates that the preference of these two types is clearly different within the same neurons (Fig. 4 e). This distribution of rigor-KIF5 and -KIF17 is consistent with the destinations of tailless motors. Although motor domains of KIF5 and KIF17 had a property to bind to MTs in the IS, this is not always the case because rigor-KIF2 did not localize on MTs in the IS, as shown by the simultaneous expression of rigor-KIF2 and rigor-KIF5 in hippocampal neurons (Fig. 4 f). We further examined the axon preference of the rigor-KIF5 by using immunoelectron microscopy. Fig. 4 (g–i) shows biased binding of rigor-KIF5 to the IS (Fig. 4 g, arrow). These results suggest that KIF5 motor domain, as default, has a preference to the MTs in the IS and tailless KIF5 is preferentially sorted to axons.

Bottom Line: Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons.We found that KIF5 has a preference to the microtubules in the initial segment of axon.These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Tokyo, Japan 113-0033.

ABSTRACT
Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1-YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

Show MeSH
Related in: MedlinePlus